Summary of Work: This project includes those endeavors in which the mass spectrometry workgroup collaborates with other groups, both inside and outside the Institute to solve problems of mutual interest. The major focuses of these projects are: 1) structure determination of unknown compounds; 2) identification and/or confirmation of biological pathways; 3) quantitation; and 4) development of strategies for the structure determination of biologically important compounds. Current major collaborative projects under way include: 1) identification of ligands for orphan receptors (C. Weinberger, LRDT) and; 2) quantitation of arachidonic acid metabolites (D. Zeldin, LPP). Quantitation of Arachidonic Acid Metabolites - The most sensitive and accurate method for quantitation of eicosanoids in physiological samples is by selected ion monitoring (SIM) under negative ion chemical ionization (NICI) mass spectrometry. We are currently quantifying epoxy- and dihydroxy- eicosatrienoic acids. One project that involves quantitation of epoxyeicosanoids is a study of the cardiac cytochrome P450 arachidonic acid epoxygenase pathway (IRA). In this study a new P450 monoxygenase, designated CYP2J3, has been sequenced and cloned. Abundant expression of CYP2J3 in rat heart and liver has been demonstrated. The principal arachidonic acid oxidation products have been identified as 14,15- 11,12- and 8,9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid. In perfused, ischemic rat hearts, addition of 11,12-epoxyeicosatrienoic acid resulted in a significant 1.6 fold improvement in recovery of cardiac contractility while neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraenoic acid showed any effect. Additional projects involving quantitation of arachidonic acid epoxygenase metabolites include characterization of the enzyme pathways of mammalian CYP2J subfamily of P450 monooxygenases. The recombinant proteins of this family were found to possess distinct enzymological properties. The distribution of the proteins in mammalian tissues has been investigated as well as their oxidation products in the specific tissues.